Antioxidants – an overview• Antioxidants are molecules capable of reducing the causes or effects of oxidative stress• Oxidative stress can be caused by environmental factors, disease, infection, inflammation, aging (ROS production)• ROS or “reactive oxygen species” include free radicals and other oxygenated molecules resulting from these factors• The body produces some endogenous antioxidants, but dietary antioxidants may provide additional line of defense• Flavonoids & other polyphenolics, Vitamins C & E, and carotenoids are the most common dietary antioxidants• Many herbs and botanicals also contain antioxidants

Resources:Gordon, M. H., “Dietary Antioxidants in Disease Prevention,” Natural Product Reports (1996) 13: 265-273; Pietta, P.-G., “Flavonoids as Antioxidants”, Journal of Natural Products (2000) 63: 1035-1042; Scalbert, A., Johnson, I.T., Saltmarsh, M. “Polyphenols: antioxidants and beyond,” American Journal of Clinical Nutrition (2005) 81: 215S-217S; Huang, D., Ou, B., Prior, R. “The Chemistry Behind Antioxidant Capacity Assays”, J. Agric. Food Chem. (2005) 53:1841-56.

Sources of antioxidants in the dietSources of antioxidants in the diet

Sources of antioxidants in the diet:Sources of antioxidants in the diet:

Polyphenols,Polyphenols, carotenoidscarotenoids & vitamins& vitamins

• Red wine (tannins, resveratrol, flavonoids)• Cranberries & blueberries (flavonoids & tannins)• Strawberries (ellagic acid, ellagitannins)• Tea (EGCG & other catechins, tannins)• Chocolate (catechins)• Onions (quercetin)• Spinach & leafy greens (lutein & zeaxanthin)• Eggs (lutein)• Citrus fruits (Vitamin C)• Plant oils (Vitamin E & omega-3)

OH

HO

Lutein, a carotenoid

Not all natural plant antioxidants are phenolics...

• Derived from 40 carbon isoprenoid chain precursor (phytoene) through the mevalonate pathway

• Conjugation gives the molecule high antioxidant capacity and ability to absorb harmful UV light

• Role in plant: carotenoids act as light-harvesting pigments, protect against photo-damage by scavenging peroxyl and singlet oxygen

• In humans, carotenoids are carried in the LDL along with tocopherol• Lutein and zeaxanthin are present in the human eye (macula) and

are thought to protect the retina from oxidative stress• Other observed beneficial bioactivities may or may not be linked to

the antioxidant properties

Defining “antioxidant”

• The term “antioxidant” has many definitions• Chemical definition: “a substance that opposes oxidation

or inhibits reactions promoted by oxygen or peroxides”• Biological definition: “synthetic or natural substances that

prevent or delay deterioration of a product, or are capable of counteracting the damaging effects of oxidation in animal tissues”

• Institute of Medicine definition: “a substance that significantly decreases the adverse effects of reactive species such as ROS or RNS on normal physiological function in humans

Huang, et al, J. Agric. Food Chem. 2005, 53: 1841-1856

Radicals and ROSThe enemy: “Reactive Oxygen Species” (ROS)

are highly reactive free radicals Superoxide (O2

-.) – protonation forms .OOH Hydroxyl radical (.OH) most reactive Peroxyl radicals (.OOH,.OOR) more selective Alkoxy radicals (.OR) Peroxynitrite (ONOO-) They form as the result of stress, inflammation, and

the human body’s natural defenses in vivo, many are formed in the mitochondria, by

phagocytes and peroxisomes, and by CYP450 activities.

They target tissue, proteins, lipids and DNAAging = cumulative damage over the years

What do antioxidants do?

Prevent formation of ROS Inhibit xanthine oxidase, COX, LOX, GST

monooxygenases, chelate metals Scavenge/remove ROS before they can damage

important biomolecules Aid the human body’s natural defenses

Upregulate superoxide dismutase (O2-.), catalase

(H2O2), glutathione peroxidase (endogenous AO) Repair oxidative damage Eliminate damaged molecules Prevent mutations

Lipid oxidation: a radical mechanism

• PUFAs (R-H) are major target due to reactivity of C=C• Initiation:

X· + RH X-H + R·

ROOH RO· + HO· or 2 ROOH RO· + ROO· + H2O

• Propagation

R· + O2 ROO·

ROO· + R’H ROOH + R’·• Termination

ROO· + R’OO· ROOR’ + O2

RO· + R’· ROR’• Hydroperoxides (ROOH) also oxidize to aldehydes and ketone by-

products

In the early days of antioxidant research, lipids /LDL oxidation was considered the major health complication due to oxidative stress and first step in atherosclerosis.Now we know that most diseases of aging, including many cancers and neurodegenerative diseases are also associated with long-term oxidative stress and associated inflammation

Basic free radical scavenging: phenols form resonance-stabilized radicals

OH O. O

R.

O

O

OH O

O

OH OR

O2

O

O

-ROH

-HR

Phenoxyl radicals areusually further oxidizedto quinones

caffeic acid, a phenolic acid

O

OH

OH

OR

HO

OH

cyanidin, an "anthocyanin"

Structures of some “polyphenolic” antioxidants found in fruits, vegetables & legumes

Found in blueberries, blackberries and cranberries

O

OH

OH

OR

HO

OH

quercetin,a "flavonol"

O

Found in berries, onions,and citrus fruit

O

OH

OH

OR

HO

OH

catechins

Found in chocolate and tea

OH

HO

OH

resveratrol, a"stilbene"

Found in red wine,peanuts

O

OH

HO

OH

genistein, an"isoflavone"

O

Found in soy productsand legumes

Found in herbs, coffeeand fruits

On a molecular level, all of these compounds “absorb” harmful free radicals and chelate pro-oxidant metal ionsMost of them also modulate cellular biochemical reactions and the expression of genes and proteins associated with oxidative stress

Flavonoids as antioxidants

Flavonoids are especially effective because of structural features including:

• Conjugation to further stabilize radicals

• ortho-dihydroxysubstituted B ring allows for chelation of pro-oxidant metal ions

(Fe2+, Fe3+, Cu2+, etc.)

• -unsaturated ketone and 3-OH on C-ring

O

OH

OH

OR

HO

OH O

Despite its mythical powers, flavonoids identified in Acai are similar to those found in other fruits

Orientin = luteolin-8C-glucoside (above)Homoorientin = luteolin-6C-glucoside (below)Luteolin is a flavone. These compounds are unusual because the sugaris attached to a C instead of O, making it more difficult to hydrolyze the glycosidic linkage

O

OH

OH

OR

HO

OH

cyanidin, an"anthocyanin"

R = glucose or rutin

PACs

The “French Paradox”The “French Paradox”

In certain regions of France, the incidence of cardiovascular disease is relatively low, despite a diet high in saturated fats

ResveratrolResveratrol&&FlavonoidsFlavonoids

Flavonoids protect against effects Flavonoids protect against effects of cardiovascular diseaseof cardiovascular disease

The Zutphen Elderly Study• A large cohort of Dutch men aged 50 to 69

years were examined in 1970 and followed up for 15 years for dietary factors and incidence of disease

• Dietary intake of flavonoids correlated with reduced incidence of stroke and reduced coronary heart disease mortality (Hertog, et al, Lancet 1993)

O

OH

H

H

OH

H

HHO H

OH

H

H

H OH

HO HO

HO

O

OH

OH

OR

HO

OH O

OH

gal=galactopyranose ara = arabinofuranose

O

OH

OMe

OR

HO

OH O

O

OH

OH

OR

HO

OH O

R = gal, ara R = gal, ara, rhamno, xyl

O

OH

H

H

OH

H

HHO H

Me

rham = rhamnopyranose

O

OH

H

H

OH

H

HHO

xyl = xylose

R = gal

O

OH

OH

OR

HO

OH

R = gal, ara

O

OH

OMe

OR

HO

OH

R = gal, ara

cyanidin

quercetinmyricetin

methyl quercetin

peonidin

Antioxidants in cranberries (Vaccinium macrocarpon):Flavonol and anthocyanin glycosides

Cranberry PACsCranberry PACsare oligomers of epicatechin units thatcontain both A and B-typelinkages between units

Extracts contain oligomers up to 12 DP

These compounds have antibacterial, antitumor and antioxidant activity

HO

OH

OH

OH

OH

O

HO

OH

OH

OH

OH

O

OHHO

OH

OH

HO O

OHO

OHHO

HOO

4

8

4

6

7

84

2

Tetramer of catechin & epicatechin units

Cranberry proanthocyanidins

B-type linkage

A-type linkage

Resveratrol…Resveratrol…the fountain of youth?the fountain of youth?

• Produced by plants in response to stress

• Found in red wine, grape and cranberry juice, legumes

• Thought to contribute to the “French Paradox”

• Decreases lipoprotein oxidation leading to cardiovascular disease (early 90’s)

• Anticancer & antiinflammatory activity (1997)

• Extends lifespan through sirtuin activation, enhancing mitochondrial function (2006)

Extending lifespan: the sirtuins• Sir2 family of proteins (silent information regulator)

regulate aging & longevity in lower organisms

• NAD+-dependent protein acetylases that regulate gene silencing, DNA repair & recombination

• Sirtuins mediate life-extending effect of caloric restriction• Analogous SIRT1 gene found in mammals• SIRT1 is a key regulator of energy and metabolic

homeostasis.• May regulate apoptosis (modulation of p53 tumor

suppressor) and differentiation• Modulates adipogenesis by deactivating PPAR

triggering loss of fat, similar to caloric restriction de la Lastra & Villegas (2005) Mol. Nutr. Food Res. 49: 405-430

Abstract: • Diminished mitochondrial oxidative phosphorylation and aerobic capacity are associated with reduced longevity. We tested whether resveratrol, which is known to extend lifespan, impacts mitochondrial function and metabolic homeostasis. • Treatment of mice with resveratrol significantly increased their aerobic capacity, as evidenced by their increased running time and consumption of oxygen in muscle fibers. • Resveratrol’s effects were associated with an induction of genes for oxidative phosphorylation and mitochondrial biogenesis• A resveratrol-mediated decrease in acetylation of PGC-1 (controls mitochondrial biogenesis and function) and an increase in PGC-1 activity was observed. • This mechanism is consistent with resveratrol being a known activator of the protein deacetylase, SIRT1.• Importantly, resveratrol treatment protected mice against diet-induced-obesity and insulin resistance.

Reduction of oxidative stress in mitochondria:

Authors group antioxidant assays into two categories:1.H atom transfer reactions (HAT) – monitor rxn kinetics2.Electron transfer reactions (ET) – involve a redox rxn with the oxidant that can be monitored

Review discussesthe pros and cons of each type of antioxidant assay

Total phenolics determination: Folin-Ciocalteu reaction

• Developed by Singleton and Rossi (1965)• Measures total phenolics content of a preparation,

expressed as gallic acid equivalents (or other standard)• Total phenolic content often correlates well with

antioxidant capacity (not always however)

• Yellow reagent prepared from solution of Na2WO4 and Na2MoO4 dihydrates, and is thought to contain heteropolyphospho-tungstates-molybdates (PMoW11O40)4-

• Phenolate anions reduce Mo(VI) to Mo (V) by electron-transfer reaction, producing a blue color that is quantified at 750 nm – species thought to be (PMoW11O40)4-?

Evaluation of antioxidant efficacy: Selected antioxidant assays

1) Free – radical scavenging (DPPH or TEAC assay)

2) Lipid oxidation / peroxidation assay (TBARS)

3) LDL oxidation assays

4) ORAC assay

5) Assays measuring redox reactions of iron (FRAP)

6) Cellular antioxidant assay (CAA)

Resources: excerpts from:Yan, X., Murphy, B.T., Hammond, G.B., Vinson, J. A., Neto, C.C. “Antioxidant activities and antitumor

screening of extracts from cranberry fruit” J. Agric. Food Chem. (2002) 50: 5844-5849.Seeram, N. and Nair, M. “Inhibition of lipid peroxidation and structure-activity related studies of the dietary

constituents anthocyanins, anthocyanidins and catechins” J. Agric. Food Chem (2002) 50: 5308-5312.Vinson, J. et al, “Vitamins and especially flavonoids in common beverages are powerful in vitro antioxidants

which enrich LDL and increase their oxidative resistance after ex vivo spiking in human plasma” (1999) J. Agric. Food Chem. 47: 2502-2504.

Wolfe, K. and Liu, R.H. “Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants, Foods, and Dietary Supplements” J. Agric. Food Chem. (2007) 55, 8896–8907.

General free radical-scavenging ability:General free radical-scavenging ability:the DPPH Assaythe DPPH Assay

Antioxidant activity of extracts and compounds can beevaluated by a general radical-scavenging assay that

predicts ability to quench OH., ROO. and other ROS.

antioxidant Violet ------------------> Yellow

• Radical-scavenging activity is determined by measuring degree of absorbance quenching for varying sample concentrations

• Activity expressed as EC50 = concentration required to quench 50% of DPPH radical

NO2

O2N

O2N

(Ph)2-N-N

DPPH: 2,2-diphenyl-1-picrylhydrazyl radical

max = 517nm.H

Cranberry flavonoids were more effective Cranberry flavonoids were more effective free radical scavengers than Vitamin Efree radical scavengers than Vitamin E

ECEC5050 for DPPH assay for DPPH assay

Compound Compound ( (g/mL)g/mL) ((M)M)

myricetin-3-arabinosidemyricetin-3-arabinoside 7.8 7.8 17.0 17.0

quercetin-3-galactosidequercetin-3-galactoside 9.6 9.6 20.7 20.7

cyanidin-3-galactosidecyanidin-3-galactoside 3.5 3.5 7.7 7.7

Trolox/Vit E (standard)Trolox/Vit E (standard) 7.5 7.5 30.0 30.0 Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A. and Neto, C. C.

J. Agric. Food Chem (2002) 50: 5844-5849

CompoundCompound ICIC5050 ( (M)M)

myricetin-3-arabinosidemyricetin-3-arabinoside 3.5 3.5

quercetin-3-galactosidequercetin-3-galactoside 4.3 4.3

cyanidin-3-galactosidecyanidin-3-galactoside 1.5 1.5

Vitamin E (standard)Vitamin E (standard) 2.4 2.4

Inhibition of low-density lipoprotein oxidation Inhibition of low-density lipoprotein oxidation in vitroin vitro by by cranberry flavonoids is comparable to Vitamin Ecranberry flavonoids is comparable to Vitamin E

Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A. and Neto, C. C.J. Agric. Food Chem (2002) 50: 5844-5849

Lipid peroxidation as the targetTBARS assay for LDL oxidation: Joe Vinson, Univ. of Scranton

• Used to test flavonoids and other dietary antioxidants for ability to prevent lipoprotein oxidation

• LDL / VLDL are reacted with varying concentrations of antioxidant in the presence of cupric ion (Cu2+) to induce formation of oxidation

products from unsaturated FA

• After 6 hrs @ 37oC, thiobarbituric acid (TBA) added

• Formation of conjugated diene oxidation products measured by fluorescence % inhibition = control - native LDL – sample fluorescence x 100/control fluorescence

Oxidation products oflipids or LDL react with TBA to form colored adducts that can be detected by absorbance or fluorescence

How do these popular antioxidantsand beverages stack up inprotecting plasma lipids?

J. Agric. Food Chem (2004) 52:5843-48

TBARS assay was used todetermine antioxidant activity

Pro-oxidant or anti-oxidant?

• Cu(II)-initiated oxidation of LDL produces decomposition products like hexanal

• Their production is measured using GC and correlated with level of oxidation

• Complication: Cu(II)-catalyzed oxidation can be promoted by the presence of excess antioxidants (e.g. tocopherols, which donate an e- to produce Cu(I), and the resulting radical reacts with the lipids)

Protecting against iron-induced lipid oxidation

ORAC assay• ORAC (oxygen radical absorbance capacity)

assay is used extensively to compare antioxidant activities of foods, beverages, and antioxidant capacity of human blood samples in a clinical setting.

• ORAC is based on the inhibition of peroxyl-radical-induced oxidation initiated by thermal decomposition of azo-compounds such as 2,2’-azobis(2-amidino-propane) dihydrochloride (AAPH)

• Free radical damage to a fluorescent probe is quantified by measuring the change in its fluorescence intensity.

• The inhibition of free radical damage by an antioxidant is assessed by comparing probe fluorescence in presence or absence of the antioxidant.

• Grandfathers of ORAC: method was developed by Dr. Guohua Cao in 1992. In 1995, Dr. Cao joined Dr. Ronald L. Prior's group at Jean Mayer USDA Human Nutrition Research Center on Aging to develop a semi-automated ORAC assay.

ORAC values are expressed as mmoles of Trolox equivalents per unit mass or volumeTrolox = water-soluble Vitamin E analog

Use of ORAC to compare antioxidant power of foods or change in plasma antioxidant capacity over time in response to a treatment

Source: Brunswick labs (http://brunswicklabs.com/app_orac.shtml)

Fe induced formation of hydroxyl radical

Fe2+ + H2O2 Fe3+ + OH_ + OH

Fenton Reaction:

Hydroxyl radical OH, very reactive, t1/2 ca 10-9 s

Consequences: Oxidative DNA damage, protein modification, lipid peroxidation, etc. Even small amounts of ferrous iron in the body can lead to the production of a large number of hydroxyl radicals.

Ascorbate(AscH-) + Fe3+ → •Asc- + Fe2+

H. J. H. Fenton, J. Chem. Soc., Trans. 1894: 899

Fluorescent sensing of iron-induced oxidation in cells (Guo, 2010)

Fig. 3. The RS-BE sensor can detect iron/H2O2-induced oxidative stress in live cells. Confocal fluorescence images of live human SH-SY5Y cells with the treatment of RS-BE/Fe/ H2O2 (scale bar 10 µm). (a) DIC; (b) the cells incubated with 10 µM RS-BE for 30 min; (c) the cells were then incubated with 10 µM Fe(8-HQ) for 30 min; (d) and (e) the cells were further treated with 100 µM H2O2 for 10 and 25 min, respectively, (f) Integrated emission (547-703 nm)  intensity of (a), (b), (c), (d) and (e) images.

FRAP and similar assays measure ability to reduce Fe3+ Fe2+

• Benzie & Strain (1999) Methods in Enzymology• FRAP reagent contains ferric (Fe3+ ) tripyridyl

triazine complex • Reduction to ferrous (Fe2+) tripyridyl triazine

forms a blue complex• Reducing capacity of compounds or mixtures

are measured based on change in absorbance at 593 nm

Cellular Antioxidant Activity (CAA) assay Can antioxidant activity be measured directly inside cells?

• Dye precursor DCFH diffuses into the cell

• Cells treated with ABAP, azo compound that forms peroxyl radicals

• Peroxyl radicals oxidize dye to fluorescent form

• Cells are treated with antioxidants

• If AO makes it into cell and scavenges the radicals, fluorescence decreases

A study of oxidative stress

• Canadian researchers found that wild blueberries decreased damage to brain cells caused by stroke-like conditions– Sweeney, M. et al: Nutritional Neuroscience, 2002

• Collaborative study (Neto & Sweeney) investigated whether cranberry could also prevent this type of damage

Ischemic Stroke

Hypoglycemia & Hypoxia

Loss of cell homeostasis, cell death by two paths:necrosis and apoptosis (Martin, 1998)

Reactive oxygen species[O2

-., H2O2]

Oxidative damage, cell death (Chan, 2001)

Reperfusion

An in vitro model that can predict stroke damage

After incubation, samples are collected and analyzed for markers of necrosis (LDH) or apoptosis (caspase-3)

Cerebellar granule neurons from neonatal rat brain are cultured at 37oC 7-10 days

Control6 hr

Oxygen Glucose Deprivation 6 hr

Simulated ischemia

1 mM H2O2

6 hrReperfusion(oxidative stress)

Cranberryphenolic extract

Flavonolsand/or Anthos

PACs

Cranberryphenolicextract

PACs

Flavonolsand/orAnthos

Cranberryphenolicextract

Flavonolsand/or Anthos

PACsn = 6/group

Percent decrease in both types of stroke-induced damageat the highest dosage level of crude extract (0.3 mg/mL)

Whole Cranberry Extract Protected Neurons from Stress-Induced Death

Necrosis Apoptosisoxidative stress (reperfusion) 42.7% 36.5%oxygen/glucose deprivation (simulated ischemia) 48.5% 50.0%

Neto, C., Lamoureaux, T., Kondo, M., Sweeney-Nixon, M., Solomon, F., MacKinnon, S. Phenolics in Foods and Natural Health Products Symposium, ACS Books (2005).

Which compounds were most effective?Which compounds were most effective?

O

OH

OH

OR

HO

OH R = gal, ara

cyanidin

* P < 0.05

O

OH

OH

OR

HO

OH O

quercetin

Indications from tissue culture model:

• Whole cranberry extract can inhibit brain cell death in vitro by up to 50%

• The anthocyanins contributed most strongly to protection, particularly in the oxidative stress model

• The whole cranberry extract is more protective against apoptosis than the fractions, suggesting that the phenolics work synergistically and that the mechanism is more than just free-radical scavenging

But what happens in vivo???

6-week feeding study (Sweeney):Rats on a diet of feed supplemented with commercial cranberry

powder (equivalent to 2.8 cups/day human dosage)

n = 5 - 7

Possible treatment effect, but not statistically significant

What happens in vivo?

• Anthocyanins do get into the brain (ACS, JAFC, 2005) but the bioavailability of other flavonoids to brain is unknown

• Most anthocyanins get broken down to smaller phenolics• Plasma levels of intact anthocyanins (nmol/L) are too low

for radical-scavenging but may be sufficient to modulate cell signalling and gene expressiona

• Other mechanisms: bilberry anthocyanins (Vaccinium myrtillus) decrease capillary fragility and permeability

• Antiinflammatory properties: inhibition of COX-2 and prostacyclin activity may relax blood vessels

• Combination of antioxidant and other effects

aMilbury, et al, 2010, Journal of Nutrition